IDH1 and IDH2 mutant AML cases tend to cluster based on their DNA methylation profiles

(A) Heatmap representation of a correlation matrix in which each patient’s DNA methylation profile is correlated with that of the other patients in the dataset. Patients are ordered according to the unsupervised analysis (hierarchical clustering) results, so that highly correlated patients are located next to each other. Parallel bars on the right of the heatmap have been used to indicate, from left to right: cluster membership, IDH1 mutational status (Green: WT, Dark red: Mutant), IDH2 mutational status (Green: WT, Dark red: Mutant) and combined IDH1/2 mutational status (Green: WT, Dark red: Mutant). (B) Heatmap representation of a correlation matrix in which each patient’s gene expression profile is correlated with that of the other patients in the dataset. Patients are ordered according to the unsupervised analysis (hierarchical clustering) results, so that highly correlated patients are located next to each other. Parallel bars on the right of the heatmap have been used to indicate, from left to right: IDH1 mutational status (Green: WT, Dark red: Mutant), IDH2 mutational status (Green: WT, Dark red: Mutant) and combined IDH1/2 mutational status (Green: WT, Dark red: Mutant). (See also and ).

(A) 293T cells were transiently transfected with empty vector, wild-type or R132H mutant IDH1, or wild-type or R172K mutant IDH2. After 3 days, cells were lysed and assessed for IDH1 expression levels by Western blot, and then re-probed for IDH2. β-actin antibody was used as a control. (B) Cells transfected in parallel to those lysed in (A) were extracted for intracellular metabolites. Metabolites were then derivatized with MTBSTFA and analyzed by GC-MS. Shown is the quantitation of 2HG signal intensities relative to the intrasample glutamate signals for a representative experiment. (C) Global DNA methylation levels in cells were analyzed 3 days following transfection by immunofluorescence using antibody against 5-methylcytosine. Quantification of fluorescence intensities from one experiment is shown. Data is representative of three independent experiments. (D) 32D cells were transduced with empty retroviral vector or with wild-type or R172K mutant IDH2, selected in 2.5 µg/ml puromycin for 7 days, and then lysed to confirm stable expression of IDH2. Tubulin antibody was used as a control. (E) Cells were extracted for their intracellular metabolites which were then derivatized with MTBSTFA and analyzed by GC-MS. Shown are representative gas chromatographs from wild-type and mutant IDH2 expressing cells depicting the derivatized metabolites eluting between 31.3 and 33.5 min, including 4-oxoproline (4-oxo Pro), glutamate (Glu), and 2HG. Metabolite abundance refers to GC-MS signal intensity. (F) DNA was extracted from cells with stable wild-type or mutant IDH2 expression, and global DNA methylation levels were measured by slot blot using antibody against 5-methylcytosine. Relative intensity of signals of three independent experiments was quantified. Error bars: +/− SD for triplicate experiments. (See also )

(A) 293T cells were transiently transfected with FLAG-tagged TET2 in the absence or presence of wild-type or R132H mutant IDH1. Three days following transfection, global levels of 5-methylcytosine hydroxylation were analyzed by immunofluorescence using antibody against 5-hydroxy-methylcytosine (5-OH-MeC). Representative images from mock-transfected, TET2-transfected, TET2 + IDH1 WT co-transfected, and TET2 + IDH1 R132H co-transfected cells are shown. Scale bar: 100 µM. (B) Transfected cells were analyzed by flow cytometry and gated as TET2 positive or negative by FLAG antibody. Representative gating is shown. Intensities of 5-OH-methylcytosine staining within the TET2 positive and negative populations are shown as histogram overlays. Data in (A) and (B) are representative of three independent experiments. (See also )

(A) 32D cells retrovirally transduced with empty vector, IDH2 WT, IDH2 R140Q, IDH2 R172K or three independent shRNAs against mouse TET2 were analyzed for C-Kit expression by flow cytometry. Intensities of fluorescence signals are depicted as histograms. (B) Primary mouse bone marrow cells were retrovirally transduced with MIGR1 vector, IDH2 WT, IDH2 R140Q, or two shRNAs against mouse TET2. GFP-positive cells were sorted and expanded in methylcellulose media for 14 days Cells were analyzed for Mac-1 and C-Kit expression by flow cytometry. (C) Cells treated as in (B) were analyzed for Mac-1 and Gr-1 expression by flow cytometry. (D) Murine primary bone marrow cells were retrovirally transduced with MIGR1 vector, IDH2 WT, IDH2 R140Q, or two shRNAs against mouse TET2. Cells were grown in liquid culture for 5 days ex-vivo and assessed for the percentage of LSK cells out of the total lineage-negative, GFP-positive cell population. (See also )